Quantitative reverse transcription-PCR-HPLC for nerve growth factor mRNA using a deletion RNA as an internal standard.
We have developed a convenient method for the routine measurement of the absolute amount of nerve growth factor (NGF) mRNA in tissue samples. The method consists of RNA extraction, amplification by reverse transcription-PCR and detection by high-performance liquid chromatography. The addition of a deletion mutant RNA to tissue samples as an internal standard enabled correction for RNA recovery during extraction, and the target mRNA and the internal standard were both amplified with the same PCR primers. The conditions were optimized so that the procedure was conducted in the region where the calibration curve was linear, thereby allowing high reproducibility and reliability. The method was applied to the measurement of NGF mRNA in tissues such as skin and skeletal muscle, where the levels are too low to be easily detected by Northern blotting analysis: skin, 14.1 +/- 4.6 fg/mg tissue and skeletal muscle, 11.0 +/- 2.2 fg/mg tissue (mean +/- SD, n = 10). The coefficient of variation of this method was less than 2.8%. This approach should also be applicable to the routine assay of the absolute amount of other mRNAs present at low levels in tissues.
Shimizu
H
H
Eisai Tsukuba Research Laboratories, Ibaraki, Japan.
Kuroki
J
J
Ogura
H
H
Yamanishi
Y
Y
Arakawa
Y
Y
D000818:Animals; D015152:Blotting, Northern; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005260:Female; D017353:Gene Deletion; D006624:Hippocampus / Q000737:chemistry; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D018482:Muscle, Skeletal / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results; D012867:Skin / Q000737:chemistry
D009414:Nerve Growth Factors; D012333:RNA, Messenger